New methods for confocal imaging of infection threads in crop and model legumes

Angus E Rae,Rosemary G White,Vivien Rolland,Ulrike Mathesius

doi:10.1186/s13007-021-00725-6

Abstract

BackgroundThe formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections.ResultsWe have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized.ConclusionsOur methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Highlights

The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process
The bacteria remain inside the matrix, and outside the plant cell wall, the wall of the infection thread being continuous with the root hair cell wall, and containing similar cell wall components including cellulose, esterified and non-esterified pectin as well as xyloglucans [5]
Exocytosis of cell wall material into the infection chamber and growing infection thread is crucial for its elongation, and this requires a complex composed of the exocyst unit EXO70H4, that interacts with vapyrin and the putative E3 ligase LUMPY INFECTIONS (LIN) [8]

Summary

Introduction

The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. In most legumes, including the model legumes Medicago truncatula and Lotus japonicus, nodulation begins when a bacterial cell makes contact with the tip of a growing root hair cell, and induces the formation of an infection thread. Exocytosis of cell wall material into the infection chamber and growing infection thread is crucial for its elongation, and this requires a complex composed of the exocyst unit EXO70H4, that interacts with vapyrin and the putative E3 ligase LUMPY INFECTIONS (LIN) [8]

Methods

Results

Discussion

Conclusion